One of the most fundamental problems in molecular neuroscience and cell biology is the proper assembly of signal-transducing membranes including the transport and sorting of protein components. A major cause of neurodegenerative and other inherited disorders is the improper localization of receptors and other signaling or transport proteins. The goal of this study is to identify proteins that interact with rhodopsin during transport and those involved in the biogenesis of disk membranes in the outer segment of rod cells, and then determine the molecular mechanisms by which the molecular interactions of rhodopsin with other proteins lead to formation of healthy photoreceptor disk membranes. This work will further the understanding of the mechanisms of neurodegenerative disorders caused by improper trafficking of receptors and other membrane proteins. The focus of the proposed research is to understand protein-protein interactions that are defective when rhodopsin lacks the proper structure at its carboxy-terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will use powerful mouse knock-in models that my co-workers and I have developed, as well as new models proposed herein. In Aim 1, we will identify proteins that interact with rhodopsin's carboxy-terminus to mediate proper transport and disk membrane assembly through affinity-capture experiments using retinal extracts from homozygote rhodopsin mutants with defective carboxyl-termini knock-in animals. In Aim 2, we will characterize, first in vitro, then in vivo, a mutant rhodopsin, Ter349Glu, containing a carboxyl-terminal extension that causes one of the most severe forms of rhodopsin-mediated autosomal dominant retinitis pigmentosa. In Aim 3, we will develop a new tool, human rhodopsin fused to photoactivatable green fluorescent protein that is followed by a repeat of rhodopsin's carboxyl terminus (rho-paGFP- 1D4). This construct will be used in two distinct ways: first, we will test the hypothesis that an unobstructed rhodopsin carboxy-terminus is sufficient to form proper outer segments in healthy rods in knock-in animals. Second, we will study the role of specific protein-protein interactions in rhodopsin trafficking after photoactivation of GFP, enabling us to track the movement of subpopulations of rhodopsin in cells for the first time. This sets the stage for in vivo trafficking studies in the future. PUBLIC HEALTH RELEVANCE: The focus of this study is to understand protein-protein interactions that are defective when the dim light photoreceptor rhodopsin lacks the proper structure at its carboxy- terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will study the role of rhodopsin in proper rod cell formation and degeneration, and monitor its trafficking to better understand these processes.